The goal of this proposal is to determine how membrane trafficking during asymmetric cell division controls a Notch signaling pathway-mediated cell fate switch. Understanding how fundamental cell biological mechanisms are mobilized to establish a specific context to regulate the Notch signaling pathway will provide insights into how dysregulation of these mechanisms in stem and progenitor cells contributes to human diseases, including cancer. One such context is the progenitor cells of the Drosophila adult peripheral nervous system (PNS), which provide a powerful model for the study of binary cell fate decisions based on the activation or inhibition of Notch activity. We have generated exciting new in vivo reagents to exploit this system using state of the art imaging techniques. We will build on the genetic framework our lab and others have established that a specific subset of conserved membrane trafficking regulators are required for correct Notch- mediated cell fate assignments after asymmetric cell division. These membrane regulators do not control Notch signaling during tissue patterning and lateral inhibition, rather they appear to target Sanpodo, a four pass transmembrane protein expressed exclusively in asymmetrically dividing cells. Previously, we demonstrated that Sanpodo promotes Notch activity to confer correct cell fates after asymmetric cell division in the adult peripheral nervous system. In preliminary studies, we developed Sanpodo-GFP, an in vivo reporter of Sanpodo protein dynamics, which faithfully recapitulates Sanpodo function in Notch signaling. Furthermore, through genetic and biochemical studies, we demonstrate that the N-terminal region of Sanpodo, which contains a previously uncharacterized and evolutionarily conserved motif, is critical for Sanpodo's function. In live imaging studies we show that key trafficking regulators are required for Sanpodo sorting to two discrete membrane domains within minutes after asymmetric progenitor cell mitosis. We hypothesize that evolutionarily conserved vesicle trafficking regulators function to establish these membrane domains to promote Notch signaling in one daughter cell, and to inhibit Notch signaling in the other daughter cell. Our lab is poised to dissect the evolutionarily conserved molecular, cellular, and genetic mechanisms underlying the establishment asymmetric cell fate decisions by our unique ability to combine molecular modeling, biochemical analysis, and live cell imaging of progenitor cell behavior in the intact animal, and propose the following specific aims: Aim 1: To determine the mechanism of Sanpodo function in controlling Notch signaling at the plasma membrane in asymmetrically dividing cells. Aim 2: To determine the roles of conserved regulators of vesicle trafficking in regulating Notch signaling. Elucidating the mechanisms of spatial-temporal control of signaling in cell fate decisions is critical to our understanding of how vertebrate neurogenesis is regulated and how failure of these mechanisms leads to disease states. PUBLIC HEALTH RELEVANCE: Elucidating the mechanisms of spatial-temporal control of signaling in cell fate decisions is critical to our understanding of how vertebrate neurogenesis is regulated and how failure of these mechanisms leads to disease states.